A prominent role of LncRNA H19 in H. pylori CagA induced DNA damage response and cell malignancy

Helicobacter pylori (H. pylori), together with its CagA, has been implicated in causing DNA damage, cell cycle arrest, apoptosis, and the development of gastric cancer. Although lncRNA H19 is abundantly expressed in gastric cancer and functions as a pro-oncogene, it remains unclear whether lncRNA H19 contributes to the oncogenic process of H. pylori CagA. This study investigates the role of H19 in the DNA damage response and malignancy induced by H. pylori. It was observed that cells infected with CagA+ H. pylori strain (GZ7/cagA) showed significantly higher H19 expression, resulting in increased γH2A.X and p-ATM expression and decreased p53 and Rad51 expression. Faster cell migration and invasion was also observed, which was reversed by H19 knockdown in H. pylori. YWHAZ was identified as an H19 target protein, and its expression was increased in H19 knockdown cells. GZ7/cagA infection responded to the increased YWHAZ expression induced by H19 knockdown. In addition, H19 knockdown stimulated cells to enter the G2-phase and attenuated the effect of GZ7/cagA infection on the cellular S-phase barrier. The results suggest that H. pylori CagA can upregulate H19 expression, participate in the DNA damage response and promote cell migration and invasion, and possibly affect cell cycle arrest via regulation of YWHAZ.


H. pylori strains, cell lines, and infection methods
The human gastric epithelial adenocarcinoma cell lines AGS and SGC7901, GZ7/cagA (we identified CagA + H. pylori wild strain, GenBank accession ID: KR154737.1)and GZ7/ΔcagA (cagA gene knockout from the GZ7/ cagA was constructed and identified by Sangon Biotech, Shanghai, China) were cultured and infected according to the methods described in previous research work 21 .

RNA FISH
RNA fluorescence in situ hybridisation (FISH) technique was used to detect the subcellular localisation of H19.AGS and SGC-7901 cells were grown on a glass slide and then infected with GZ7/ΔcagA or GZ7/cagA at the multiplicity of infection (MOI) of 60.The experiment was performed according to the instructions of the FISH kit (GenePharma Inc, China).Once the cells reached 80% confluence, they were rinsed with PBS and then treated with 4% formaldehyde at room temperature for fixation.The cells were then washed again with PBS, permeabilized with Triton X-100, dehydrated through a series of ethanol washes and hybridised overnight with a digoxigenin-labelled H19 probe.Confocal microscopy (Carl Zeiss LSM 880, Germany) was utilized to examine the RNA FISH signal, which was detected by incubation with an Alexa Fluor 647-labelled anti-digoxigenin antibody.

RT-qPCR assay
Cells from the study groups were harvested and total RNA was extracted using TRIzol reagent (Invitrogen, USA).The Transcriptor First Strand cDNA Synthesis Kit (Roche, USA) was used to reverse transcribe the RNA into cDNA.FastStart Universal SYBR Green I Master (ROX, Roche, USA) and CFX Connect PCR System (Bio-Rad, USA) were used for qRT-PCR according to the manufacturer's instructions.GAPDH was used as internal reference.The expression of H19 and YWHAZ was determined by the 2−∆∆Ct method and normalised to GAPDH.The sequence of all primers (Sangon, Biotech, China) used in this study is shown in Table 1.
Table 1.The primers for real-time reverse transcription-polymerase chain reaction.

RNA pull-down and mass spectrometry identification
Sense and antisense H19 strands were obtained from the pcDNA3.1-H19plasmid by PCR technique.Biotinlabelled RNAs of H19 were synthesised in vitro using the Biotin RNA Labelling Mix and T7 RNA polymerase (cat.no.20163; Thermo Fisher Scientific, Inc.USA).The RNA pull-down experiment was performed using the PierceTM Magnetic RNA-Protein Pull-Down kit (Cat.No. 20164; Thermo Fisher Scientific, Inc. USA) according to the manufacturer's instructions.Mass spectrometry (Sangon, Biotech, China) was used to determine the protein-binding complexes of lncRNA from the RNA pull-down experiment.

Cell cycle analysis
AGS cells were first transfected with si-H19 and then infected with H. pylori.The cells were then fixed in 75% ice cold ethanol overnight.After two washes with PBS, the propidium iodide (PI)-treated cells were thoroughly rinsed with PBS according to the manufacturer's instructions of the cell cycle analysis kit (KGA512, Keygen, China).The cell cycle was detected by flow cytometry (FACS verse, BD Bioscience, USA).

Statistical analysis
Experiments were performed three times, and the data analysis was performed using

H19 expression and cytoplasmic localisation were increased in H. pylori infected cells
To investigate the effect of H. pylori infection on H19 expression and the potential role of CagA in this process, the AGS and SGC7901 cells were infected with GZ7/ΔcagA and GZ7/cagA strains (MOI 60:1) for 24 h.Total RNA and protein were extracted from the infected cells.First, the absence of CagA protein expression in the GZ7/ ΔcagA strain was verified by Western blot analysis (Fig. 1A).Subsequently, qRT-PCR analysis showed an increase in H19 expression in AGS and SGC7901 cells infected with GZ7/ΔcagA and GZ7/cagA strains.Interestingly, the upregulation of H19 was more pronounced in the GZ7/cagA infection group compared to the GZ7/ΔcagA infection group (Fig. 1B).Furthermore, FISH analysis revealed a significant increase in H19 localisation in the cytoplasm after infection with GZ7/ΔcagA and GZ7/cagA strains compared to the control group with H19 being more abundant in the cytoplasm of GZ7/cagA infected cells (Fig. 1C).Thus, CagA was found to enhance H19 expression and potentially increase its cytoplasmic localisation (Supplementary Information).

H19 participates in the regulation of the DNA damage response
Evidence has indicated that H. pylori can disrupt DNA repair pathways and other responses to DNA damage 25 .In addition, previous research has shown that H. pylori can induce H19 overexpression.Therefore, it is necessary to investigate the potential involvement of H19 overexpression in the DNA damage response.After transfection of the pcDNA3.1-H19into the AGS and SGC7901 cells, qRT-PCR findings results indicated a significant increase in H19 expression in the pcDNA3.1-H19transfected cells (ov-H19) compared to the pcDNA3.1 empty vector transfected cells (pcDNA3.1;Fig. 2A).Western blot analysis was performed to investigate the effects of increased H19 expression.The results showed an up-regulation of γH2A.X and p-ATM proteins, which are crucial in the context of DNA damage.In contrast, the expression of p53 and Rad51, two proteins involved in DNA repair, was observed to be less frequent (Fig. 2B).Immunofluorescence detection of γH2A.X protein in the ov-H19 group revealed a significant increase in its average fluorescence level (Fig. 2C).These results indicated that overexpression of H19 plays an important role in the regulation of the DNA damage repair response.

H19 can participate in the DNA damage response generated by H. pylori CagA
To gain insight into the role of H19 in the DNA damage response induced by H. pylori CagA, AGS and SGC-7901 cells were transfected with si-H19 and subsequently infected with GZ7/cagA and GZ7/ΔcagA strains.The results of qRT-PCR indicated that H19 knockdown could significantly reduce the increased H19 expression caused by H. pylori infection (Fig. 3A).Western blot analysis was performed to determine the levels of γH2A.X, p-ATM, p53, and Rad51 proteins in the cells from each group.Knockdown of H19 was found to reverse the upregulated expression of γH2A.X, p-ATM, and decreased expression of p53 protein caused by H. pylori infections.However, the expression of Rad51 protein significantly reduced by si-H19 transfection and H. pylori infection, thus suggesting that H19 knockdown may minimise the DNA damage response caused by H. pylori infection but has minimal effect on Rad51-related DNA repair (Fig. 3B).The average fluorescence intensity of γH2A.X was assessed using the immunofluorescence assay to confirm that H19 knockdown could potentially reduce DNA damage caused by H. pylori infection (Fig. 3C).Overall, H19 appears to play a role in the DNA damage response mediated by H. pylori and CagA.

H19 is upregulated in gastric cancer and associated with poor prognosis
To identify H19s potentially involved in gastric cancer progression, we analysed H19 expression profiles using the GEPIA II databases.The results showed that H19 was overexpressed in gastric cancer tissues compared to adjacent normal tissues (Fig. 4A).We further investigated whether H19 expression correlated with the outcome of gastric cancer patients.Kaplan-Meier survival estimates showed that high H19 expression in gastric cancer tissues was significantly associated with worse overall survival (p = 0.061, log-rank test) (Fig. 4B).Analysis of H19 expression in gastric cancer patients with different clinicopathological characteristics showed that high or low H19 expression was positively correlated with pathological parameters such as T stage and AJCC stage (all p < 0.05, Table 2).The study also investigated the effects of H19 on invasion, migration and proliferation of GC cells.The results of Transwell and wound healing assays showed that the cells transfected with ov-H19 (pcDNA3.1-H19)exhibited a significant increase in wound healing, migration and invasion activity compared to the cells transfected with empty vector (pcDNA3.1).(Fig. 4C,D).These results indicated that increased expression of H19 may play a pivotal role in the pathogenesis of gastric cancer and the malignant outcome of patients with gastric cancer.

H19 knockdown reduces H. pylori-induced cell proliferation, migration, and invasion
To gain a more complete understanding of the cellular behaviour of H. pylori-infected cells, transfection of AGS and SGC-7901 cells with si-H19 was performed prior to infection with GZ7/ΔcagA and GZ7/cagA strains.RTCA assay of cell proliferation showed that si-H19 transfection significantly decreased the proliferation ability of cells compared to the normal control group, and H19 knockdown significantly inhibited cell proliferation induced by GZ7/ΔcagA and GZ7/cagA infection (Fig. 5A).Furthermore, H19 knockdown also reduced the healing ability of GZ7/ΔcagA and GZ7/cagA infected cells based on the results of the wound healing assay (Fig. 5B).Transwell assay showed that H19 knockdown significantly decreased the invasion and migration ability of GZ7/ΔcagA and GZ7/cagA infected cells (Fig. 5C).Furthermore, GZ7/cagA-infected cells showed more pronounced effects compared to GZ7/ΔcagA-infected cells.These results demonstrated that H19 knockdown reduced H. pyloriinduced cell proliferation, migration and invasion, indicating its potential involvement in these processes.

H19-interacting protein screening and identification
The present results suggest that H19 may play a critical role in H. pylori-associated GC.To investigate the possible interaction of H19 with specific proteins, biotin-labelled antisense-H19 magnetic beads and biotinlabelled sense-H19 magnetic beads were used.The beads were used in RNA pull-down assays in conjunction  for mass spectrometry analysis, a total of 20 differentially expressed proteins bound to H19 were identified in the GZ7/cagA-infected group (sense-H19-H.pylori) compared to the uninfected group (sense-H19; Table 3).A literature search of 20 H19 target proteins revealed that the YWHAZ protein is linked with cell cycle control and tumorigenesis.YWHAZ, also known as 14-3-3ζ, encodes an adapter protein from the 14-3-3 family.It affects a number of signalling pathways, including cell proliferation, cell cycle, apoptosis, migration, and invasion, and is critical in tumorigenesis 26,27 .Therefore, we postulate that H. pylori may be implicated in carcinogenesis by suppressing YWHAZ expression through H19.

H19 can affect the distribution of cell cycle upon H. pylori infection via YWHAZ
To investigate the effect of H. pylori and H19 on YWHAZ expression, pcDNA3.1-H19was introduced into AGS cells and allowed to grow for 48 h.It was observed that overexpression of H19 could lead to the downregulation of YWHAZ, as confirmed by RT-qPCR and Western blotting analysis (Fig. 6A).Furthermore, AGS cells were transfected with si-H19 and subsequently infected with GZ7/cagA (MOI 60:1) for 24 h to analyse YWHAZ expression.The results showed that YWHAZ expression was higher in cells transfected with si-H19.However, infection with GZ7/cagA decreased the expression of YWHAZ.Interestingly, GZ7/cagA infection counteracted the upregulation of YWHAZ expression induced by si-H19.These findings imply that YWHAZ, as an H19 binding protein, may play an important role in the pathogenic process associated with H. pylori infection (Fig. 6B).
Since YWHAZ is closely linked to the cell cycle, the effect of H19 knockdown on the cell cycle distribution of H. pylori-infected cells was investigated.The results showed that transfection of si-H19 into the cells increased the number of cells entering the S phase and G2 compared to the control group.In addition, H. pylori infection of si-H19-transfected cells increased their entry into the G2 phase, suggesting that knockdown of H19 improved the cell cycle block caused by H. pylori infection.These results further confirmed that H19 could regulate cell cycle distribution by H. pylori by using its target protein YWHAZ (Fig. 6C).

Discussion
GC is one of the leading causes of cancer death worldwide and is associated with high morbidity.Numerous studies have shown that GC is strongly associated with atrophic gastritis due to H. pylori infection 28 .H. pylori secretes numerous virulence factors that have the potential to disrupt intracellular signalling pathways in the host, thereby facilitating the onset of neoplastic transformation.Among these factors, cytotoxin-associated gene A (CagA), vacuolating cytotoxin A (VacA) and outer membrane proteins (OMPs) have been implicated in the development of gastric cancer 29 .All H. pylori isolates secrete vacuolating cytotoxin A (VacA) via the type V secretion system.VacA has several effects on the host cell, including cell vacuolization, alteration of mitochondrial membrane permeability, inhibition of T-lymphocyte activation and proliferation, and activation of cell signalling 30 .H. pylori attach to the gastric epithelial cell and transfer the CagA protein into the cell via the OMPs, which is associated with gastric cancer 2 .CagA-induced cellular changes relevant to cancer pathogenesis include inhibition of apoptosis, stimulation of cell proliferation, degradation of the p53 tumour suppressor and double-strand DNA breaks.It is thought that cagA-positive strains are associated with more severe inflammation, higher levels of atrophy and a greater likelihood of progression to gastric adenocarcinoma than cagA-negative strains 31 .To date, there has been relatively little progress in determining the pathogenesis of GC by H. pylori CagA 32 .H19 is widely expressed in GC tissues and plays an important role in the progression of GC through multiple pathways 33 .The results of the present study showed a significant increase in H19 expression and cytoplasmic localisation in H. pylori infected cells, consistent with the observations of Zhang et al. 10 .Another notable finding was that the GZ7/cagA strain showed a pronounced upregulation of H19 expression compared to the GZ7/∆cagA strain.This suggests a possible link between the upregulation of H19 and the pathogenicity factor CagA in H. pylori infection.Research has shown that the localisation of lncRNAs within cells can significantly affect the stability and translation of mRNA in the cytoplasm, allowing them to perform specific functions 34 .According to a study by Yang et al. 35 , H19 was found to be predominantly located in the cytoplasm of keratin-forming cells and may exert pro-inflammatory effects by activating the NF-κB signalling pathway.Therefore, it was postulated that H19 overexpression induced by H. pylori infection may also act predominantly in the cytoplasm and be involved in its pathogenic process.
DDR involves activating ATM and ATR, phosphorylating H2AX histones at Ser 139 (γ-H2AX), activating sensors Chk1 and Chk2, and loading the HR core repair protein RAD51, which activates p53 36 .p53 plays a central role in responding to DNA damage and determines the outcome of the DNA damage checkpoint response by regulating cell cycle arrest and apoptosis 37 .Two main mechanisms are used for DSB repair: non-homologous end joining (NHEJ) and homologous recombination (HR).In the HR pathway, RAD51 catalyses this process by promoting strand-invasion of a resected 3ʹ single-stranded DNA (ssDNA) end into the homologous repair template; mutational inactivation or abnormal RAD51 production can cause genomic phenotypic instability, which in turn increases the risk of spontaneous DNA damage and cancer 38 .H19 has previously been proposed to regulate the DNA damage response; mass spectrometry analysis of potential H19-interacting proteins revealed several DNA damage response and repair molecules directly associated with H19, but the underlying mechanisms remain to be defined 39,40 .The current study found that H19 overexpression significantly increased the expression Table 3. Differentially expressed proteins of sense-H19-H.pylori/sense-H19.Compared with the control group, "+" marks the up-regulated proteins; "−" marks the down-regulated proteins.could potentially increase DNA damage while limiting repair capacity, which is consistent with the findings of Wang et al. 41 , who identified the ability of H19 to cause DNA damage in hepatocellular carcinoma cells.H. pylori infection is associated with increased DNA damage.CagA may act as a critical regulator of DNA damage repair in H. pylori infection, altering the balance between DNA damage and repair, leading to genomic instability and cancer development 42,43 .In view of this, RNA interference methods were used to generate AGS and SGC7901 cell models with reduced levels of H19 expression, which were subsequently infected with GZ7/ΔcagA and GZ7/ cagA strains, respectively, to detect the expression of γH2A.X, p-ATM, P53, and Rad51.The results of Western blotting and immunofluorescence assays suggested that knockdown of H19 expression could effectively inhibit the H. pylori CagA-induced DNA damage response in GC cells.However, the effect of CagA-regulated H19 expression on DNA damage repair might not be related to the Rad51 repair mechanism.The results suggest that the increased expression of H19 driven by H. pylori CagA may play an important role in the DNA damage response induced by H. pylori.
In the current study, it was observed that H19 overexpression could significantly enhance the invasion and migration of GC cells, which was consistent with the previous finding that H19 could promote invasion and migration via involvement in the miR-138/E2F2 axis in GC, as proposed by Yu et al. 44 .Further investigation showed that H19 knockdown in GC cells reduced proliferation, invasion, and migration capabilities.Interestingly, H19 knockdown effectively reduced cell proliferation, invasion, and migration generated by GZ7/ΔcagA and GZ7/cagA infection.Notably, the effect was more pronounced in the GZ7/cagA-infected group than in the GZ7/∆cagA-infected group.The results suggest that H. pylori CagA infection can modulate GC cell proliferation, invasion and migration by altering H19 expression.
It is importance to identify the specific proteins that interact with the lncRNA H19.To achieve this RNA pull-down, combined mass spectrometry was used to identify H19-binding proteins associated with H. pylori infection.This can facilitate the study of individual proteins that interact with H19 and may play a role in the development of GC associated with H. pylori infection.A total of 20 differentially expressed H19-binding proteins were identified in the GZ7/cagA infected and GZ7/cagA uninfected groups.One of these proteins, YWHAZ, has been reported to be closely associated with various signalling transduction pathways and the cell cycle.It plays a critical role in DNA damage repair and carcinogenesis 45,46 .A more detailed analysis of the effects of H. pylori infection and H19 on YWHAZ expression was performed.The results showed that GZ7/cagA infection decreased YWHAZ expression, which is consistent with the observed reduction in YWHAZ expression in cells overexpressing H19.In contrast, YWHAZ expression was upregulated in H19 knockdown cells, and GZ7/cagA infection could respond to the high YWHAZ expression caused by H19 knockdown.This suggests that YWHAZ may play a role in the development of H. pylori-related pathogenesis as a protein that binds to H19.
H. pylori infection can induce genetic instability by damaging DNA, leading to cell cycle arrest and apoptosis in gastric epithelial cells.The results of further experiments confirmed that cells infected with GZ7/cagA were significantly blocked in the S phase.These findings were consistent with the results of Gao's 47 on the induction of G1/S arrest in AGS cells upon H. pylori infection.H19 knockdown cells showed an increase in S phase and G2, consistent with the observations made by Ping Liu et al. 48in their study on glioma cells after knockdown of H19.Further analysis showed that H19 knockdown alleviated GZ7/cagA infection-induced S phase arrest and facilitated cell entry into the G2 phase, thereby improving cell cycle progression.However, further experiments are required to confirm the role of YWHAZ in this process.

Conclusions
In this current study, H. pylori infection can lead to the upregulation of H19 gene expression, which subsequently affects the DNA damage response and facilitates the invasion and migration of GC cells.However, the knockdown of H19 can significantly inhibit the DNA damage response and the migratory and invasive ability of GC cells induced by H. pylori infection.In addition, H19 was found to be involved in cell cycle arrest induced by H. pylori infection through the regulation of its binding protein YWHAZ.The CagA+ strains induced a stronger biological response in this process than the CagA knockdown strains.Thus, by regulating the expression of H19, H. pylori CagA can effectively modulate the DNA damage repair response and promote proliferation, migration, and invasion of GC cells.
GGC CAA GAC GCC AGG R-TCC TCT GTC CTC GCC GTC AC YWHAZ F-ATT GAA CAA AAG ACG GAA R-CAG CCA AGT AAC GGT AGT A GAPDH F-AGA AGG CTG GGG CTC ATT TG R-AGG GGC CAT CCA CAG TCT TC
We examined the differential expression and Kaplan-Meier (K-M) overall survival (OS) of H19 in the GEPIA II database (http:// gepia2.cancer-pku.cn/).The survival results were presented as hazard ratios (HRs) and p values from a log-rank test.The p value cut-off was 0.01.The correlation of H19 expression with clinicopathological features of gastric cancer was further investigated using The Cancer Genome Atlas (TCGA) dataset (https:// xenab rowser.net/ datap ages/), using the χ 2 test.Student's t-test or one-way analysis of variance (ANOVA) was used to determine statistical significance.The level of statistical significance was set at p < 0.05.

Table 2 .
The relationship between the expression level of H19 and the clinicopathological parameters of GC.